Adenoviral vectors encoding an antibody fused to a CD4 extracellular domain

ABSTRACT

Genetically modified cell implant comprising an exogenous nucleotide sequence coding for all or part of an antibody, method for the preparation of such an implant and its therapeutic use for the treatment or prevention of an acquired disease. The invention also concerns an adenoviral vector for the expression of one or more proteins capable of forming a multimer, viral particles and cells containing the adenoviral vector, a pharmaceutical composition and its therapeutic use.

The present invention relates to a novel type of implant and its use forthe treatment and prevention of cancer or of AIDS. More particularly,its subject is an implant comprising genetically modified cells capableof expressing and of secreting specific antibodies recognizing cancercells or infected cells so as to inhibit at least partially theirdivision or propagation as well as the production of viral particles inthe infected cells. The present invention also relates to an adenoviralvector capable of directing the expression of a multimeric protein ofinterest as well as an antibody or one of its derivatives.

The possibility of treatments of human diseases by gene therapy has gonewithin a few years from the theoretical considerations stage to that ofclinical applications. The first procedure applied to man was thusinitiated in the United States in September 1990 on a geneticallyimmunodeficient patient because of a mutation affecting the geneencoding Adenine Deaminase (ADA). The relative success of this firstexperimentation encouraged the development of new gene therapyprocedures for various genetic or acquired diseases. Those currentlyunder experimentation consist, for the most part, in transferring exvivo the therapeutic gene into the patient's cells, for example the stemcells of the hematopoietic line, and then reinfusing these correctedcells into the patient. It is therefore a nonreversible, cumbersometechnology which carries the risk of reimplanting transformed cells.

More recently initiated, the neo-organ technology makes it possible toovercome the major disadvantages of the conventional gene therapyprocedures. It is based on the reimplantation, in the patient, of anartificial structure which may be called “implant” and comprising livingcells, which are real “micro-factories” which make it possible todeliver the therapeutic molecule of interest in vivo and continuously.

More precisely, this artificial structure consists of living cellspreviously transduced by a viral vector carrying the therapeutic gene,which are included in a collagen gel coating a backbone of syntheticfibers of a biocompatible material (PTFE, polytetrafluoroethylene orGore-Tex™). This gel also contains an angiogenic growth factor (bFGF,basic Fibroblast Growth Factor). After its reimplantation in the animal,the neo-organ is generally vascularized within a few days by virtue ofthe angiogenic and trophic properties of bFGF. It then develops into anautonomous structure, provided with a connective, sometimes innerated,tissue and linked to the bloodstream into which the therapeuticmolecules are poured.

The possibility of using neo-organs for gene therapy has already beenraised in several scientific articles as well as in internationalapplication WO 92/15676. However, the technology disclosed in the priorart documents deals with only the treatment of monogenic geneticdiseases resulting from the defective and innate expression of a singlegene, and has consequently been used only for the secretion of monomerictherapeutic molecules such as factor IX, α₁-antitrypsin, ADA,erythropoietin (EPO) and β-glucuronidase. Up until now, this technologyhas not been suited to the secretion of more complex therapeuticmolecules such as antibodies.

It has now been found that an implant of fibroblasts, geneticallymodified by a retroviral vector for the expression of the heavy andlight chains of an anti-HIV antibody, once reimplanted in a mouse, iscapable of continuously secreting into the bloodstream a large quantityof functional antibodies recognizing the infected cells carrying, attheir surface, the antigen against which it is directed. The presentinvention is based on the fact that a fibroblast is capable of producingroughly stoichiometric quantities of heavy and light chains of anantibody capable of then associating into a tetramer to form afunctional molecule. It offers the possibility of treating, byimmunotherapy, acquired diseases and especially AIDS and cancer, twodiseases whose complexity, seriousness as well as the absence of reallysatisfactory treatments, justify the development of novel technologies,such as that which is the subject of the present invention.

The present invention also provides adenoviral vectors capable ofdirecting the expression of multimeric molecules of interest as well asof antibodies and derivatives thereof. They can be used to produce animmunotoxin directed against the HIV virus and to induce the selectivedestruction of infected cells.

Accordingly, the subject of the present invention is:

(1) an implant of genetically modified cells comprising an exogenousnucleotide sequence encoding all or part of an antibody, the saidexogenous nucleotide sequence being placed under the control of theelements necessary for its expression and for the secretion of the saidantibody, and

(2) a recombinant adenoviral vector comprising an exogenous nucleotidesequence encoding all or part of one or more protein(s) of interestcapable of forming a multimer in a host cell; the said exogenousnucleotide sequence being placed under the control of the elementsnecessary for its expression in the said host cell.

For the purposes of the present invention, an implant designates any setof genetically modified living cells, as defined below and intended tobe implanted in the human or animal body. Most particularly preferred isthe case where the cells are attached to an extracelluar matrix, thewhole forming a biocompatible and vascularizable structure. The matrixis preferably composed of collagen. However, other materials may be usedwithin the framework of the present invention as long as they arebiocompatible. It comprises especially (1) a biocompatible support suchas synthetic fibers PTFE (polytetrafluoroethylene or Gore-Tex) coatedwith a collagen film so as to allow cell adhesion (2), a collagen gel inwhich the cells inside the implant are included and (3) an angiogenicagent promoting vascularization in the host. The term implant is ageneric term which includes especially neo-organs and organoids.

Moreover, this may also involve encapsulated implants, that is to sayincluded in a membrane of controlled porosity preventing especially thepassage of cells (cells of the implant and cells of the host's immunesystem) but allowing the diffusion of the therapeutic molecule,nutrients and waste.

The term “genetically modified cell” refers to a cell havingincorporated exogenous genetic material. The latter may be inserted intothe genome of the cell or be present in episome form either in thecytoplasm or in the cell nucleus. The technology for introducing anexogenous genetic material into a cell is conventional and accessible topersons skilled in the art. In this regard, numerous vectors have beendeveloped and are widely described in basic molecular biology manualsaccessible to persons skilled in the art.

The genetically modified cells in use within the framework of thepresent invention comprise especially an exogenous nucleotide sequence.The latter may be a natural sequence (already present in the genome ofthe host cell) or a heterologous sequence, but it will have beenintroduced into the host cells by genetic engineering techniques (andtherefore exogenously). Most particularly preferred is a sequenceencoding a product which is not normally expressed therein or, if it is,at physiologically low concentrations. In accordance with the aimspursued by the present invention, the exogenous nucleotide sequenceencodes all or part of an antibody. An antibody is a protein(immunoglobulin) normally produced by the B lymphocytes and whichrecognizes a specific foreign antigen and triggers the immune response.A native antibody is a tetramer composed of four protein chains: twolight (L) chains and two heavy (H for heavy) chains associated with eachother via disulfide bridges. The light chain consists of a variableregion (V_(L)) at the N-terminal position and a constant region (C_(L))at the C-terminal position whereas the heavy chain comprises from the Nto the C-terminal a variable region (V_(H)) followed by three constantregions (C_(H1), C_(H2) and C_(H3)). The corresponding regions of thelight and heavy chains associate to form distinct domains. The variabledomain, formed by the association of the variable regions of the lightand heavy chains of an immunoglobulin, is responsible for recognizingthe corresponding antigen. The constant domains exert effector functionsinvolved in the progress of the immune response.

For the purposes of the present invention, the two heavy and lightchains may be identical (native antibodies). In this context, anexogenous nucleotide sequence is used which encodes a heavy chain and alight chain which will associate into a tetramer after their synthesis.However, a sequence may also be used which encodes only part of anantibody so as to produce, preferably, a fragment Fab (ab for antigenbinding) or F(ab′)₂, Fc (c for crystallizable) or scFv (sc for singlechain and v for variable). Such fragments are described in detail inimmunology manuals such as Immunology (third edition, 1993, Roitt,Brostoff and Male, ed Gambli, Mosby) and are schematically representedin FIG. 1. As regards more specifically the scFv fragment, it may beobtained from a sequence encoding a V_(L) region followed by a V_(H)region with optionally a spacer (of 1 to 10 neutral amino acid residueswhich are not very bulky) between the V_(L) and V_(H) sequences.

It is also possible to generate a chimeric (or hybrid) antibody derivedfrom the fusion of sequences of diverse origins (species or types ofantibody). In particular, it is possible to include or exchange constantregions derived from antibodies of different isotopes so as to confernew properties on the chimeric antibody, for example an enhancement ofthe cytotoxic reaction. This may also be a humanized antibody combiningat least part of the variable regions of a mouse antibody and theconstant regions of a human antibody. It is also possible to fuse one ormore variable and/or constant regions or region parts of any origin, forexample derived from light/or heavy chains in the form of a single-chainmolecule.

Finally, another approach consists in producing a bispecific antibodycomprising two variable domains, for example a domain recognizing anantigen carried by an infected or a tumor cell and the other a structurefor activation of the immune response. This makes it possible toincrease the activity of the killer cells in contact with the tumor orwith the infected cell.

It goes without saying that an antibody in use in the present inventionmay have a sequence which is slightly different from the native sequenceof an antibody. In practice, the common criterion for characterizing anantibody is its function, that is to say its capacity to bindspecifically to the antigen against which it is directed. Numeroustechniques which appear in general immunology manuals make it possibleto demonstrate an antibody function, for example the ELISA, Western orfluorescence techniques. The invention extends to an antibody whosesequence has a degree of homology with the native sequence(s) (in thecase of a chimeric antibody) greater than 70%, advantageously greaterthan 80%, preferably greater than 90% and, most preferably, greater than95%. Such an analogue may be obtained by mutation, deletion,substitution and/or addition of one or more nucleotide(s) of thecorresponding sequence(s).

In accordance with the aims pursued by the present invention, it ispreferable to use an antibody directed against a tumor antigen or anepitope specific for an infectious and pathogenic microrganism,especially a virus and more particularly the HIV virus and,advantageously, an antigen strongly represented at the surface of thetarget cell. This type of antibody is widely described in theliterature. There may be mentioned especially:

-   -   the human monoclonal antibody 2F5 (Buchacher et al., 1992,        Vaccines, 92, 191-195) recognizing a continuous (ELDKWAS) (SEQ        ID NO: 21) and highly conserved epitope of the transmembrane        glycoprotein gp41 of the HIV-1 envelope molecule,    -   the murine monoclonal antibody 17-1-A (Sun et al., 1987, Proc.        Natl. Acad. Sci. USA, 84, 214-218) recognizing the GA733        glycoprotein present at the surface of the human colorectal        carcinoma cells,    -   an antibody directed against the protein MUC-1, and    -   an antibody directed against the E6 or E7 protein of the HPV        virus (Human Papillomavirus) especially type 16 or 18.

Within the framework of the present invention, the nucleotide sequencesencoding an antibody in use within the framework of the presentinvention may be obtained by any conventional technique in use in thefield of genetic engineering, such as PCR (Polymerase Chain Reaction),cloning and chemical synthesis. Purely as a guide, the sequencesencoding the heavy and light chains of an antibody may be cloned by PCRusing the degenerate oligonucleotides recognizing the conservedsequences found at the 5′ and 3′ ends of most immunoglobulin genes(Persson et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 2432-2436; Burtonet al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10134-10137). The antibodyfunction of the expression product is then checked in relation to aspecific antigen as indicated above.

Another approach, which is moreover preferred, consists in using anantibody which is modified especially by a toxic substance or animmunopotentiating protein. This specific embodiment makes it possibleto destroy in vivo, by a local chemotherapy (toxic substance), thetarget cell (cancer cell or infected cell) which carries, at itssurface, the specific antigen against which the antibody part isdirected or to enhance the immune reaction with respect to it(immunopotentiating substance). In the context of the toxic substance,it may be advantageous to choose antibodies which may be endocytosed bythe target cell. It goes without saying that the corresponding sequencesmay be obtained by any conventional technique in the state of the art.

The term “toxic substance” refers to a molecule having a degradationactivity drastically inhibiting cell growth or inducing cell death. Thismay be a molecule which is toxic by itself or indirectly, for example aprotein catalyzing the synthesis of a toxic substance. These moleculesmay be derived from plants, animals or microorganisms. Of course, thetoxic function may be fulfilled by a native toxic substance (as found innature) or an analog thereof, which may be conventionally obtained bymutation, deletion, substitution and/or addition of one or morenucleotide(s) of the native sequence. Among the preferred toxicsubstances, there may be mentioned a ribonuclease, ricin, diphtheriatoxin, cholera toxin, herpes simplex virus type 1 thymidine kinase(TK-HSV-1), cytosine deaminase from Escherichia coli or from a yeast ofthe genus Saccharomyces and the exotoxin from Pseudomonas. To illustratean immunopotentiating protein (whose function is to improve the hostorganism's immune reaction toward the target cell), there may bementioned the CD4 protein, the high-affinity receptor for the HIV-1virus or an Fc receptor for IgG(FcγR). Its coupling to an antibodydirected against an HIV virus antigen or a tumor antigen will make itpossible, consequently, to generate a hybrid molecule having a ligandrecognizing a killer cell and a ligand recognizing the target cell so asto promote its elimination more efficiently. In this context, a hybridmolecule may be used which is obtained from the fusion between ananti-HIV antibody and FcγR or between the extracellular domain of theCD4 molecule and an anti-CD3 antibody. However, these examples are notlimiting and such immunopotentiating proteins are known to personsskilled in the art.

Advantageously, the toxic function is provided by a ribonuclease whichmay be of prokaryotic or eukaryotic origin. Among those which may beused within the framework of the present invention, there may bementioned colicin E6, cloacin from Escherichia coli, nuclease fromStaphylococcus, birnase from Bacillus intermedius and nuclease fromBacillus amyloliquefaciens, also designated by the name barnase, whosesequence is disclosed in Hartley (1988, J. Mol. Biol., 202, 913-915).However, the use of human angiogenin is most particularly preferred(Saxena et al., 1991, J. Biol. Chem., 266, 21208-21214; Saxena et al.,1992, J. Biol. Chem., 267, 21982-21986).

According to another variant, the toxic function may be exerted byTK-HSV-1. It exhibits a greater affinity, compared with the mammalian TKenzyme, for certain nucleoside analogs such as acyclovir and ganciclovirand it converts them to nucleotide precursors which are toxic for thecell. Consequently, their incorporation into the DNA of replicatingcells makes it possible to kill specifically dividing cells, such ascancer cells, by a toxic effect and/or by a proximity effect(“bystander” effect).

According to another embodiment of the invention, an attenuated analogmay be used which also exhibits a toxic function but to a lesser degreecompared with the native toxic substance. Any mutant having anattenuated degradation activity may be used within the framework of theinvention. In this context, an attenuated mutant of a ribonuclease maybe used which exhibits an activity attenuated by a factor of 10 to 10⁶or better still 10 to 10⁵ and, most preferably, 10² to 10⁴ compared tothe native ribonuclease from which it is derived. This variant is basedon the high toxicity of the ribonucleases to cellular RNAs, which makesthe molecular construction stages difficult. By way of examples, theremay be mentioned the attenuated mutants of barnase K27A (Mossakowska etal., 1989, Biochemistry, 28, 3843-3850) and K27A, L89F (Natsoulis andBoeke, 1991, Nature, 352, 1632-1635). The nuclease activity may beevaluated in accordance with the method described by Shapiro et al.(1987, Proc. Natl. Acad. Sci. USA, 84, 8783-8787). Of course, it ispossible to measure it by other techniques too, as indicated in Example2.

A particularly preferred construction consists in including thenucleotide sequence encoding the said toxic or immunopotentiatingsubstance in 5′ or in 3′ of the nucleotide sequence encoding all or partof an antibody. There is especially preferred the case where it isintroduced downstream of the sequence encoding the heavy chain of anantibody, the said chain being deleted of the stop codon for translationand the fusion taking place in the correct reading frame. The fusion oftwo sequences operably constitutes a conventional molecular biologytechnique accessible to persons skilled in the art. Moreover, it ispossible to include, at the level of the fusion, a binding sequencecapable of being cleaved inside the target cell in order to release thetoxin. In this context, the term “exogenous nucleotide sequence” refersto a sequence encoding all or part of an antibody optionally fused tothe said substance.

Of course, the said exogenous nucleotide sequence is placed under thecontrol of elements which are necessary for its expression. “Elementswhich are necessary” is understood to mean all the elements which arenecessary for its transcription into messenger RNA (mRNA) and for thetranslation of the latter into protein. Among the elements which arenecessary for the transcription, the promoter is of particularimportance. In general, a promoter will be used which is functional in aeukaryotic, and especially human, cell. This may be a constitutivepromoter or a regulatable promoter and it may be isolated from any geneof eukaryotic or viral origin. Moreover, a promoter in use in thepresent invention may be modified so as to contain regulatory sequences,such as “enhancer” type activating sequences. Alternatively, a promoterderived from immunoglobulin genes may be used when it is desired totarget a lymphocytic host cell. Nevertheless, it will be preferable touse a constitutive promoter allowing expression in a large number ofcell types and especially a promoter of a housekeeping gene such as thepromoter of the TK-HSV-1 gene, the adenoviral promoter E1A, MLP (forMajor Late promoter), the murine or human PGK (phosphoglycerate kinase)promoter, the promoter of the rat β-actin (ACT) gene, the HPRT(Hypoxantyl Phosphoribosyl Transferase) promoter, the HMG(Hydroxymethyl-Glutaryl coenzyme-A) promoter, the RSV (Rous SarcomaVirus) promoter, the SV40 virus (Simian Virus) early promoter or theDHFR (Dihydrofolate Reductase) promoter. As a guide, when the nucleotidesequence is incorporated into a retroviral vector, the 5′ LTR may beused as promoter. However, it is most particularly preferable to use aninternal nonretroviral promoter, such as those specified earlier.

The exogenous nucleotide sequence may, in addition, contain otherelements contributing to its expression both at the level oftranscription and translation, especially an intron sequence bordered byappropriate splicing signals, a nuclear localization sequence, asequence for initiation of translation, the elements for termination oftranscription (polyadenylation signal), and/or a sequence encoding asecretory signal. The said sequence may be homologous, that is to sayderived from the gene encoding the antibody in question, orheterologous, that is to say derived from any gene encoding a precursorof a secreted expression product. The choice of such elements is wideand accessible to persons skilled in the art.

For the purposes of the present invention, the exogenous nucleotidesequence provided with the elements necessary for its expression isintroduced into a host cell to give a genetically modified cell. All theprocedures which make it possible to introduce a nucleic acid into acell may be used, such as for example precipitation with calciumphosphate, DEAE dextran technique, direct injection of nucleic acid intothe host cell, the bombardment of gold microparticles covered withnucleic acid or the use of liposomes or of cationic lipids. However,within the framework of the present invention, the exogenous nucleotidesequence is preferably inserted into an expression vector. Inparticular, it may be of the plasmid type or derived from an animalvirus and especially a retrovirus, an adenovirus, anadenovirus-associated virus or a herpes virus. However, the use of anintegrative vector is preferred. The choice of such a vector is wide andthe techniques for cloning into the vector selected are accessible topersons skilled in the art. Likewise, the process to be used to generateinfectious viral particles is known.

A first vector which is particularly appropriate for the presentinvention is an adenoviral vector (see below).

According to another, also advantageous, alternative, a retroviralvector is used. The numerous vectors described in the literature may beused within the framework of the present invention and especially thosederived from the Moloney murine leukemia virus (MoMuLV) or from theFriend's virus (FrMuLV). In general, a retroviral vector in use in thepresent invention is deleted of all or part of the viral genes gag, poland/or env and comprises a 5′ LTR, an encapsidation region and a 3′ LTR.The exogenous nucleotide sequence is inserted preferably downstream ofthe encapsidation region. The propagation of such a vector requires theuse of complementation lines described in the prior art, such as thelines CRE, GP+E-86, PG13, Psi Env-am-12, pA317 and psi-CRIP.

According to a preferred embodiment and as regards producing an antibodywhich is other than a single chain (comprising for example two heavy andlight protein chains), the use of a dicistronic vector allowing thesynthesis of two translational products from a single mRNA is preferred.The initiation of translation of the second translational product ispreferably provided by an IRES site (for Internal Ribosome Entry Site,that is to say an internal site for entry of the ribosomes). A number ofIRES sites have so far been identified and there may be mentioned thatof the poliomyelitis virus (Pelletier et al., 1988, Mol. Cell. Biol., 8,1103-1112), of EMCV (Encephalomyocarditis Virus) (Jang et al., J.Virol., 1988, 62, 2636-2643) or those described in internationalapplication WO 93/03143. But other IRES sites may also be used. Thistype of construction may be appropriate for any vector in use within theframework of the invention.

One of the preferred vectors within the framework of the presentinvention is a retroviral vector which comprises from 5′ to 3′:

(a) a 5′ LTR derived from a retrovirus,

(b) an encapsidation region,

(c) an exogenous nucleotide sequence comprising:

an internal promoter

a first sequence encoding the heavy chain of an antibody,

a ribosome entry initiation site,

a second sequence encoding the light chain of an antibody, and

(d) a 3′ LTR derived from a retrovirus.

Another preferred retroviral vector comprises an exogenous nucleotidesequence provided with the murine PGK promoter followed by a firstsequence encoding the extracellular I and II domains of the CD4 moleculeand a second sequence fused in phase with the first and encoding the γ3segment of the heavy chain of the antibody 2F5 (sCD4-2F5) and,optionally, a third sequence encoding human angiogenin operably linkedto the second.

It goes without saying that the order of the first, second and thirdsequences may be reversed. Moreover, as indicated above, the exogenousnucleotide sequence may comprise a sequence encoding a toxic orimmunopotentiating substance. The latter will be preferably inserteddownstream of the first sequence encoding the heavy chain of anantibody. However, the present invention is not limited to this specificembodiment.

Moreover, a vector in use within the framework of the invention may alsocontain other elements, for example, a gene encoding a selectable markerwhich makes it possible to select or identify the host cellstransfected. There may be mentioned the neo gene which confersresistance to the antibiotic G418, the dhfr gene, the CAT(chloramphenicol Acetyl Transferase) gene, the puromycin acetyltransferase (pac or PURO) gene or the gpt (xanthine guaninephosphoribosyl transferase) gene.

A genetically modified cell is preferably chosen so as to be toleratedby the immune system of the host organism in which it is envisaged tograft an implant according to the invention. In this context, a nontumorand transfectable cell is most particularly preferred. They may beautologous cells removed or derived from this host organism, but alsocells which are capable of being tolerated following an appropriatechemical or genetic treatment (it is for example possible to envisagerepressing the expression of the surface antigens normally recognized bythe host organism's immune system). It is also possible to use asyngenic cell or an allogenic cell of the same haplotype as the hostorganism as regards the major histocompatibility complex class IIantigens.

Preferably, a genetically modified cell results from the introduction ofthe exogenous nucleotide sequence into autologous fibroblasts and, inparticular, fibroblasts removed from the skin of a host organism.However, other cell types may be used, such as endothelial cells,myoblasts, lymphocytes and hepatocytes. Although not a preferredembodiment, it is also possible to use tumor cells (optionallyattenuated by radiotherapy) removed from a host organism having tumors,in order to modify their gene pool and make them capable of inhibitingor slowing down tumor progression.

Advantageously, an implant according to the invention comprises from 10⁶to 10¹², preferably from 10⁷ to 10¹¹, and most preferably from 10⁸ to10¹⁰ genetically modified cells.

The present invention also relates to a method for the preparation of animplant according to the invention in which the genetically modifiedcells and an extracellular matrix are placed in contact. Varioustechniques may be used to generate an implant according to theinvention. The procedure is preferably carried out in the followingmanner: the genetically modified cells are brought into contact with aliquid collagen solution, preferably of type I, with a biocompatiblesupport consisting, for example, of synthetic Gore-Tex fibers coatedwith collagen and with at least one angiogenic growth factor, forexample bFGF or VEGF (Vascular Endothelial Growth Factor). The whole isplaced at 37° C. so that the collagen solution forms a gel with a densemeshwork which includes the cells and then cultured for 4 to 5 days invitro so as to allow the genetically modified cells to colonize theimplant. It is desirable to carry out the last stage of culture in amedium containing at least one angiogenic factor or a combination of twoor more. In general, the techniques which make it possible to generatean implant and the culture conditions are known to persons skilled inthe art.

An implant according to the invention is intended to be transplanted ina host, animal or, preferably, human organism so as to produce atherapeutic (curative and/or preventive) effect therein. Transplanted ina laboratory animal, it will make it possible, in particular, toevaluate therapeutic procedures applicable to man. The site ofreimplantation is preferably the peritoneal or subcutaneous,intrarachidian or intraabdominal cavity.

The invention also extends to the therapeutic use of an implantaccording to the invention for the preparation of a pharmaceuticalcomposition intended more particularly for the treatment and/orprevention of an acquired disease such as cancer or an infectiousdisease caused by a pathogenic microorganism (virus, parasite orbacterium). It relates especially to the treatment:

-   -   of cancer of the uterus induced by a papillomavirus against        which an implant will be used comprising autologous fibroblasts        into which a sequence encoding an anti-HPV (in particular of        type 16 or 18) E6 or E7 antibody has been introduced,    -   of breast cancer using an anti-MUC1 antibody,    -   of AIDS using an antibody directed against an envelope        glycoprotein epitope conserved in numerous isolates,    -   of hepatitis using an antibody directed against an epitope of        the hepatitis B or C virus.

Of course, these antibodies may be modified by fusion especially toangiogenin, barnase or TK-HSV-1.

The invention also relates to a method for the treatment or preventionof acquired diseases according to which an implant according to theinvention is generated in vitro and it is transplanted into a patientrequiring such a treatment. The sites of reimplantation may be varied asmentioned above. Once the desired therapeutic effect is obtained, theimplant simply has to be surgically removed from the patient.

Naturally, the modalities of the therapeutic procedure have to bedeveloped by the clinician according to the patient and the disease tobe treated. This procedure may be subject to numerous variants such asthe number of implants according to the invention to be transplanted,the site of implantation and the type of antibody secreted as well asthe level of expression. Purely as a guide, a level of expression in thepatient's serum of at least 50 ng/ml of functional antibody,advantageously of at least 100 ng/ml, preferably of at least 200 ng/mland, most preferably, of at least 500 ng/ml, is preferred. A functionalantibody is an antibody capable of recognizing the antigen against whichit is directed. The functionality may be determined for example by ELISAor FACS. On the other hand, when an antibody fused to TK-HSV-1 is used,it is desirable to include in the therapeutic procedure theadministration of acyclovir or of ganciclovir so that its toxic effectmay be exerted.

Moreover, the present invention also relates to a recombinant adenoviralvector comprising an exogenous nucleotide sequence encoding all or partof one or more protein(s) capable of forming a multimer in a host celland, preferably, a dimer or a tetramer. For the purposes of the presentinvention, a recombinant adenoviral vector according to the inventionmay be used alone to combat an infection induced by a pathogenicorganism or the establishment/propagation of a tumor in an organism or ahost cell. According to a completely preferred embodiment, a recombinantadenoviral vector according to the invention comprises an exogenousnucleotide sequence as defined above (intended to express an antibody orone of its derivatives such as a fragment, a modified, chimeric antibodyand the like).

A recombinant adenoviral vector according to the invention is preferablyderived from a human adenovirus serotype C and, more particularly, type2, 5 or 7. However, there may also be used other adenoviruses,especially of animal (canine, bovine, murine, avian, ovine, porcine orsimian) origin or a hybrid between a variety of species. There may bementioned more particularly the canine adenovirus CAV-1 or CAV-2, theavian adenovirus DAV or the bovine adenovirus Bad type 3 (Zakharchuk etal., 1993, Arch. Virol., 128, 171-176; Spibey and Cavanagh, 1989, J.Gen. Virol., 70, 165-172; Jouvenne et al., 1987, Gene, 60, 21-28; Mittalet al., 1995, J. Gen. Virol., 76, 93-102). The general technologyrelating to adenoviruses is disclosed in Graham and Prevec (1991,Methods in Mol. Biol., Vol. 7, Gene Transfer and Expression Protocols,Ed: Murray, The Human Press Inc., p109-118).

An advantageous embodiment of the present invention consists in using avector which is defective for one or more viral function(s) which is(are) essential for replication, because of the deletion ornon-functionality of one or more viral genes encoding the said function.Such a vector, which is incapable of autonomous replication, will bepropagated in a complementation cell capable of providing en trans theearly and/or late proteins which it cannot itself produce and which arenecessary for the constitution of an infectious viral particle. Thelatter term designates a viral particle having the capacity to infect ahost cell and to cause the viral genome to penetrate therein. By way ofillustration, to propagate an adenoviral vector which is defective forthe E1 function, there will be used a complementation cell such as theline 293 capable of providing en trans all the proteins encoded by theE1 region (Graham et al., 1977, J. Gen. Virol. 36, 59-72). Of course, avector according to the invention may comprise additional deletions,especially in the nonessential E3 region so as to increase the cloningcapacities, but also in the essential E2, E4, L1-L5 regions (seeinternational application WO 94/28152). The defective functions may becomplemented with the aid of a cell line or a helper virus.

A preferred adenoviral vector according to the invention is deleted ofmost of the E1 and E3 regions and carries, in place of the E1 region, anexpression cassette comprising:

(a) a promoter, the intron of the human β-globin (BGL) gene, thesequences encoding the light chain of 2F5, the IMES site of the EMCVvirus and the heavy chain of 2F5 and then the polyadenylation site ofthe human β-globin gene, or

(b) a promoter, the intron of the human β-globin gene, the sequencesencoding the molecule sCD4-2F5 optionally fused at the C-terminus and inthe same reading frame to human angiogenin.

Among the promoters which may be envisaged within the framework of thepresent invention, there may be mentioned the adenoviral early promoterE1A, the late promoter MLP (Major Late Promoter), the murine or humanPGK (Phosphoglycerate Kinase) promoter, the SV40 virus early promoter,the RSV (Rous Sarcoma Virus) virus promoter, a promoter which isspecifically active in tumor cells and finally a promoter which isspecifically active in the infected cells.

The invention also relates to an infectious adenoviral particle as wellas to a eukaryotic host cell comprising a recombinant adenoviral vectoraccording to the invention. The said host cell is advantageously amammalian cell and, preferably, a human cell and may comprise the saidvector in a form integrated in the genome or nonintegrated (episome).This may be a primary or tumor cell of hematopoietic origin (totipotentstem cell, leukocyte, lymphocyte, monocyte or macrophage and the like),or of muscle, hepatic, epithelial or fibroblast origin.

An infectious viral particle according to the invention may be preparedaccording to any conventional technique in the state of the art (Grahamand Prevect, 1991, supra), for example, by cotransfection of a vectorand of an adenoviral fragment into an appropriate cell or by means of ahelper virus providing en trans the non-functional viral functions. Itis also possible to envisage generating the viral vector in vitro inEscherichia coli (E. coli) by ligation or homologous recombination (seefor example French Application 94 14470).

The subject of the invention is also a pharmaceutical compositioncomprising, as therapeutic or prophylactic agent, an adenoviral vector,an infectious viral particle or a eukaryotic host cell according to theinvention in combination with a pharmaceutically acceptable carrier. Thecomposition according to the invention is in particular intended for thepreventive or curative treatment of acquired diseases such as cancers,viral diseases such as AIDS, hepatitis B or C or recurrent viralinfections caused by the herpes virus.

A pharmaceutical composition according to the invention may be producedin a conventional manner. In particular, a therapeutically effectivequantity of a therapeutic or prophylactic agent is combined with acarrier such as a diluent. A composition according to the invention maybe administered locally or systemically or by aerosol. Especiallypreferred is the intramuscular, intratumor and intrapulmonaryadministration and, most particularly, intravenous injection. Theadministration may take place in a single dose or in a dose which isrepeated once or several times after a certain interval of time. Theappropriate route of administration and dosage vary according to variousparameters, for example, the individual or the disease to be treated orthe gene(s) of interest to be transferred. In particular, the viralparticles according to the invention may be formulated in the form ofdoses of between 10⁴ and 10¹⁴ pfu (plaque forming units), advantageously10⁵ and 10¹³ pfu and, preferably, 10⁶ and 10¹¹ pfu. The formulation mayalso include an adjuvant or an excipient which is acceptable from apharmaceutical point of view.

Finally, the present invention relates to the therapeutic orprophylactic use of an adenoviral vector, an infectious viral particleor a eukaryotic host cell according to the invention for the preparationof a medicament intended for the treatment of the human or animal bodyand, preferably, by gene therapy. According to a first possibility, themedicament may be administered directly in vivo (for example byintravenous injection, into an accessible tumor, into the lungs byaerosol and the like). The ex vivo approach may also be adopted whichconsists in removing cells from the patient (bone marrow stem cells,peripheral blood lymphocytes, muscle cells and the like), in infectingthem in vitro according to prior art techniques and in readministeringthem to the patient.

The invention also relates to a method for the treatment or preventionof acquired diseases according to which a therapeutically effectivequantity of a recombinant adenoviral vector, an infectious adenoviralparticle or a host cell according to the invention is administered to apatient requiring such a treatment.

The invention is illustrated, without however being limited, by thefollowing examples and with reference to the following figures:

FIG. 1 is a schematic representation of the structure of an antibody andof the F(ab) and Fc fragments.

FIG. 2 is a schematic representation of the vector pTG4370 allowing theexpression of the antibody 2F5.

FIG. 3 is a schematic representation of the vector pTG6356 allowing theexpression of the antibody 17-1-A coupled to the native barnase.

FIG. 4 is a schematic representation of the vector pTG6357 allowing theexpression of the antibody 17-1-A coupled to the attenuated barnaseK27A.

FIG. 5 is a schematic representation of the vector pTG6355 allowing theexpression of the antibody 17-1-A.

FIG. 6 is a schematic representation of the structure of the CD4membrane protein.

FIG. 7 is a representation of the scheme for the construction ofsequences encoding the hybrid molecule sCD4-2F5 using primers OTG7094,OTG7095, OTG7096 and OTG7097 (SEQ ID NOS: 13, 14, 16 and 15).

FIG. 8 is a schematic representation of the retroviral vector pTG8338allowing the expression of the hybrid molecule sCD4-2F5.

FIG. 9 is a schematic representation of the vector pTG83 73 comprisingthe sequences encoding the fusion molecule sCD4-2F5-Angiogenin.

FIG. 10 is a schematic representation of the adenoviral vector pTG8357allowing the expression of the hybrid molecule sCD4-2F5.

FIG. 11 is a schematic representation of the adenoviral vector pTG8376allowing the expression of the fusion molecule sCD4-2F5-Angiogenin.

EXAMPLES

The constructions described below are carried out according to thegeneral genetic engineering and molecular cloning techniques detailed inManiatis et al. (1989, Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) or according to the recommendations ofthe manufacturer when a commercial kit is used. As regards the repair ofthe restriction sites, the filling of the protruding 5′ ends may beperformed with the aid of the Klenow fragment of DNA polymerase ofEscherichia coli (E. Coli) and the destruction of the protruding 3′ endsin the presence of the T4 phage DNA polymerase or by treatment with S1nuclease followed by repair with Klenow. The PCR techniques are known topersons skilled in the art and are abundantly described in PCRProtocols, a guide to methods and applications (Ed: Inis, Gelfand,Sninsky and White, Academic Press, Inc.).

The cloning steps using bacterial plasmids are preferably carried out bypassage on the E. coli XL1-Blue strain (Stratagene), and those relatingto the vectors derived from the M13 phage in E. coli NM522. Themutageneses are performed using synthetic oligodeoxynucleotides with theaid of a kit of commercial origin (for example Amersham, RPN1523) andaccording to the recommendations of the manufacturer.

Example 1 Preparation of an Implant Secreting the Antibody 2F5 andIntended for an Anti-AIDS Immunotherapy

A. Construction of a Dicistronic Retroviral Vector for the Expressionand Secretion of Anti-HIV Antibody 2F5.

The vector which forms the basis of the constructions is pLXSP which isderived from pLXSN (Miller and Rosman, 1989, BioTechniques, 7, 980-988).The latter is a retroviral vector which comprises the 5′ LTR of MoMuSV(Moloney Murine Sarcoma Virus), a retroviral encapsidation region,multiple restriction sites, the neo gene for resistance to neomycinunder the control of the SV40 promoter and the MoMuLV 3′ LTR. The vectorpLXSP is obtained after, on the one hand, the replacement of theNheI-KpnI fragment of the 3′ LTR of pLXSN by an analogous fragmentobtained from the MPSV (Myelo Proliferative Sarcoma Virus) 3′ LTRisolated from the vector pMPSV.H-2K.IL-2R (Takeda et al., 1988, GrowthFactors, 1, 59-66) and, on the other hand, introduction of the puromycinresistance gene as replacement for the neo gene. The puromycin gene isobtained from pBabe Puro described in Morgenstern and Land (1990,Nucleic Acids Res., 18, 3587-3596).

The vector pLXSP is digested with EcoRI and HpaI and an EcoRI-PstIfragment (after repairing the PstI site) isolated from pKJ-1 (Adra etal., 1987, Gene, 60, 65-74) is introduced therein. This fragment carriesthe promoter of the mouse PGK gene. After ligation, the vector pTG2663is obtained. This is subjected to a digestion with the enzymes ClaI andBamHI in order to eliminate the cassette for expression of the puromycingene. After treatment with Klenow and ligation, the vector pTG2673 isobtained in which the BamHI site is reconstituted.

The vector pTG2676 is obtained by cloning the HindIII-EcoRI fragmentcomprising the cDNA encoding the light chain (LC) of the monoclonalantibody 2F5 into the plasmid Bluescript SK+ (Stratagene). It may becloned by PCR with a cDNA library derived from the mRNA of the hybridoma2F5 (Buchacher et al., 1994, AIDS Research and Human Retroviruses, 10,359-369; Katinger, 1992, Seventh Cent Gardes Conference, 299-303) usingappropriate primers complementary to the sequences surrounding the codonfor initiation of translation and the stop codon, such as the primersOTG5168 and OTG5169 (SEQ ID NO: 1 and 2). There is isolated from pTG2676an XhoI-BamHI fragment carrying the cDNA LC 2F5 which is inserted intothe vector pTG2673 previously digested with the same enzymes in order togenerate pTG4336.

The latter is linearized with the NcoI enzyme and subjected to ligationwith, on the one hand, the NcoI-EcoRI fragment obtained from pTG2677 andcarrying the cDNA encoding the heavy chain (HC) of the antibody 2F5 and,on the other hand, the EcoRI-NcoI fragment purified from pTG4369 andcarrying the EMCV IRES site. The vector pTG2677 is a pBluescript SK+ inwhich the cDNA HC 2F5 carrying the same ends has been introduced betweenthe HindIII and EcoRI sites. It was obtained by PCR on the precedinglibrary with the aid of the primers OTG5170 and OTG5171 (SEQ ID NO: 3and 4). As for the vector pTG4369, it is obtained from the cloning, alsointo pBluescript SK+, of the XbaI-ClaI fragment corresponding to theEMCV IRES (Jang et al., 1988, supra). The triple ligation generates thevector pTG4370 (FIG. 2). The cassette for expression of the puromycingene may be optionally reintroduced into the plasmid part of pTG4370 soas to facilitate the selection stages. pTG6368 is obtained.

Infectious viral particles are generated in the following manner:

The ecotropic complementation line GP+E-86 (Markowitz et al., 1988, J.Virol., 62, 1120-1124) and the target cells NIH3T3 (mouse fibroblastcells), available from ATCC, are cultured at 37° C. in the presence of5% CO₂ in DMEM medium (Dulbecco's Modified Eagle's Medium) containing10% foetal calf serum (FCS) (GibcoBRL), 1 mM Glutamine, 1% nonessentialamino acids and 40 μg/l of gentamicin (complete DMEM medium). The daybefore the transfection, the GP+E-86 cells are cultured in an amount of5×10⁵ cells per 10 cm dish. The next day, 20 μg of linearized plasmidpTG4370 and 1 μg of selection vector (for example the vector pLXSPcarrying the puromycin resistance gene) are transfected according to thetraditional calcium phosphate method. The following day (D+1), the cellsare washed according to the prior art methods and placed in a new mediumfor 48 hours, before being cultured from D+3 in selective medium (5μg/ml of puromycin).

At the end of about 2 weeks of selection, cell clones resistant to theantibiotic are visible on the dish. They are isolated according to priorart techniques and the cells resuspended in selective medium in 96-wellculture plates. The clones which are the best producers of antibodiesare selected using the ELISA method described below and the supernatantsare titrated on NIH3T3 cells. To do this, on the day before theinfection, they are inoculated at 10⁵ cells per well. The viralinfections are performed according to the conventional proceduredescribed in the literature. The titration method is the so-calledlimiting point method. The ELISA method makes it possible to titrate thequantity of functional 2F5 antibodies recognizing the target epitope(ELDKWAS) (SEQ ID NO: 21). The latter is chemically synthesized.

Briefly, a peptide solution (2 mg/ml) is diluted 2000 fold in a buffer(15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) and 100 μl are deposited at thebottom of each well of a microtiter plate and incubated at 4° C. for 16h. After extensive washes in a PBS buffer, 0.05% Tween-20, the wells aresaturated with 50 μl of 1% BSA dissolved in a PBS buffer 1 h at 37° C.After washing, 100 μl of the sample to be tested or of a standardsolution are added. The plate is incubated for 2 h at room temperatureand washed thoroughly with the PBS buffer-Tween 20. 100 μl of aperoxidase-conjugated anti-human IgG goat antibody are then added(concentration 0.8 mg/ml; Jackson Immuno Research Laboratories Inc, PA)diluted 1000 fold in PBS buffer, 1% BSA. After 2 hours at roomtemperature and extensive washes, the peroxidase enzymatic activity isrevealed by addition of 100 μl of the following preparation (0.066 MNa₂HPO₄, 0.035 M C₆H₈O₇ pH5, 0.04% ortho-phenylenediamine and 0.014%hydrogen peroxide). The reaction is stopped by 150 μl of 1 M H₂SO₄. Theabsorbance is measured at 490 nm.

The standard solution is obtained either from a commercial source (VirusTesting Systems, Houston) or from a hybridoma supernatant concentratedon Centricon. The antibody solution (1 μg/ml) is 2-fold serially dilutedin fetal calf serum. The absorbance is measured for each of thedilutions and the calibration curve is plotted (ng of antibody as afunction of the absorbance). The most productive clones are thusdetermined.

B. Preparation of the Implant

1/Removal and Culture of Primary Fibroblasts.

Skin biopsies are performed on young 2- to 3-day old BALB/C female mice.However, other mouse lines may also be suitable. After a roughmechanical dissociation, the sample is placed in 30 ml of complete DMEMmedium in the presence of 5000 units of dispase (Collaborative MedicalProducts) and of 1% collagenase (Sigma). After 2 hours at 37° C., themixture is diluted and the cells are harvested by centrifugation,carefully washed before being resuspended in RPMI 1640 medium (GibcoBRL). After about one week of culture, the primary fibroblasts areinfected with the culture supernatants of the producing clones selectedas in the preceding stage (A) according to the conventional procedure.Fibroblast cells NIH3T3 may also be reimplanted by way of a model. Theyare cultured as indicated above and infected conventionally. Thepresence of the 2F5 antibody in the supernatants of NIH3T3 cellsinfected with one of the producing clones was monitored by the ELISAtest and was evaluated at 500 ng/ml/24 h, that is to say a productivityof more than 1 μg/10⁶ cells/24 h.

2/Preparation of a Neo-Organ

The PTFE fibers previously autoclaved (Gore Inc, AZ) are first of allplaced in contact with a rat tail collagen solution (solution at 0.5mg/ml in 0.1 N acetic acid) for 2 hours under vacuum. They are thenspread at the bottom of a well (12-well plate), UV-sterilized,rehydrated with PBS buffer overnight before being treated for 2 hours atroom temperature with angiogenic factors (10 ml of PBS containing 2 μgof bFGF and 1 μg of VEGF per 100 mg of fibers approximately).

In parallel, the infected fibroblasts (primary fibroblasts or NIH3T3)are briefly trypsinized. 1.5×10⁷ cells are resuspended in 0.2 ml ofmedium and then 2 ml of the following mixture are added per well: 200 μlof 10×RPMI, 24 μl of 7.5% sodium bicarbonate, 5 μl of 1 M Hepes, 20 μlof Gentamicin, 20 μl of Glutamine, 2 μl of bFGF (10 ng/μl), 2 μl of EGF(Epidermal Growth Factor) (10 ng/μl), 1.5 ml of collagen (2 mg/ml), 12μl of NaOH (10 N) and 15 μl of H₂O. After 30 to 60 minutes of incubationat 37° C., a polymerization of the mixture is observed. The latter iscultured at 37° C. for about 4 days in the presence, for the last night,of angiogenic factors (bFGF and VEGF).

C. Reimplantation of the Implant

One or two implants are introduced into the peritoneal cavity either ofBALB/c female mice or of nude Swiss mice. One month after theirimplantation, it is checked that they have anchored to the adiposetissue of the abdominal cavity and are vascularized. Moreover, bloodsamples are collected regularly during the month following theimplantation and the assay of the 2F5 antibody by ELISA (according tothe technique described above) reveals values of the order of 20 ng/mlof serum in syngenic BALB/c mice and exceeding 100 ng/ml of serum in thenude mice. The antibody level is maintained over a period of more than 6to 7 weeks after the implantation.

The efficacy of the 2F5 antibody to inhibit the HIV viral infection isevaluated on SCID (Severe Combined Immuno Deficiency) mice. They areimmunodeficient mice possessing no T cells or mature B cells which,moreover, may be humanized by introduction of human cells or tissues.This treatment makes them injectable by HIV (Namikawa et al., 1988,Science 242, 1684-1686)

One to two neo-organs secreting the 2F5 antibody are implanted in theabdominal cavity of humanized SCID mice by intraperitoneal injection ofhuman lymphocyte cells CEM A3 (40×10⁶ cells). 3 to 5 weeks after thetransplant, the mice are challenged with the HIV virus (1000 TCID₅₀ ofHIV1 Bru isolate intravenously). The cells are recovered from the animal3 days post-infection and cultured. The cell supernatant is collected atregular time intervals and the reverse transcriptase activitydetermined. It is observed that the human cells are protected againstinfection by HIV and that the protection is maintained for the entireduration of the experiment (50 days). Indeed, the reverse transcriptaseactivity is below the detection threshold in mice which have received atransplant of the implant secreting the 2F5 antibody (behavior similarto the noninfected control). These data reflect an inhibition of thereplication of HIV in the animals producing 2F5 in their bloodstream.

Example 2 Preparation of an Implant for an Anticancer Immunotherapy

A. Construction of the Dicistronic Retroviral Vector for the Expressionand Secretion of the 17-1-A Antibody.

In the first place, the plasmid pBluescript SK+ is digested with NotIand then subjected to a treatment with the large Klenow fragment of DNApolymerase before being self-religated. The vector pTG6336 is generatedin which the NotI site has been destroyed. In parallel, the cDNAencoding the light chain of the 17-1-A antibody is isolated by PCR froma cDNA library constructed from mRNA isolated from 17-1-A hybridomacells (Sun et al., 1987, Proc. Natl. Acad. Sci. USA, 84, 214-218; Herlynet al., 1979, Proc. Natl. Acad. Sci. USA 76, 1438-1442). As a guide,this antibody is directed against an epitope of the transmembraneglycoprotein GA733-2 (Szala et al., 1990, Proc. Natl. Acad. Sci. USA,87, 3542-3546) present at the surface of human colorectal carcinomacells. The PCR uses the primers OTG6114 and OTG6115 (SEQ ID NO: 5 and 6)designed so as to introduce restriction sites facilitating subsequentcloning steps, the EcoRI and NcoI sites in 5′ and the BglII and XbaIsites in 3′ respectively. After checking on agarose gel, the PCRfragment thus generated is digested with EcoRI and XbaI and then clonedinto pTG6336 between the same sites. pTG6339 is generated.

The latter is digested with EcoRI and NcoI and ligated to the EcoRI-NcoIfragment of pTG4369 carrying the IRES site, to give pTG6343. There isintroduced into the latter the cDNA encoding the heavy 17-1-A antibodychain lacking the stop codon in the place of which there is inserted asmall spacer encoding the residues Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 22).The cDNA HC 17-1-A is obtained by PCR from the preceding cDNA libraryand using the oligonucleotides OTG6192 and OTG6194 (SEQ ID NO: 7 and 8).The insertion of the PCR fragment digested with SalI-EcoRI makes itpossible to generate pTG6346.

The latter is linearized with NotI and then ligated to the NotI fragmentcarrying the sequence encoding barnase to give pTG6347. The geneencoding barnase is obtained by PCR from a preparation of Bacillusamyloliquefaciens genomic DNA and the primers OTG5147 and OTG5148 (SEQID NO: 9 and 10). The oligonucleotides were designed so as to introducean NotI restriction site in 5′ of the codon corresponding to the firstamino acid of the mature barnase and in 3′ of the stop codon. It ischecked that the sequence of the PCR fragment thus generated is inconformity with that published in Hartley (1988, J. Mol. Biol., 202,913-915).

In parallel, the NotI fragment is inserted into an Ml 3-type vector, forexample the vector M13TG130 (Kieny et al., 1983, Gene, 26, 91-99) inwhich an NotI site has been previously introduced inside the cloningsite by site-directed mutagenesis. The modification of the restrictionsites by site-directed mutagenesis is a technique known to personsskilled in the art. The vector thus obtained is subjected to asite-directed mutagenesis with the aid of the oligonucleotide OTG5299(SEQ ID NO: 11) so as to modify the lysine residue (Lys or K) atposition 27 of the native barnase by an alanine residue (Ala or A).Next, the modified NotI fragment is isolated and introduced, as above,into the vector pTG6346 to give pTG6348.

The SalI-BglII fragment isolated from the vector pTG6347 or pTG6348 istransferred into the vector pTG2673 previously digested with XhoI andBamHI. The vectors pTG6356 and pTG6357 are obtained respectively (FIGS.3 and 4).

Moreover, a dicistronic vector is constructed which contains thesequences encoding HC 17-1-A, the EMCV IRES followed by LC 17-1-A. TheHC fragment provided with a stop codon is obtained by PCR from thevector pTG6346 with the oligonucleotides OTG6192 (SEQ ID NO: 7) andOTG6193 (SEQ ID NO: 12). This PCR fragment, which is equipped with SalIand EcorI sites at its 5′ and 3′ ends respectively, is inserted into thevector pTG6343 digested with the same enzymes to give pTG6345. TheSalI-BglII fragment of the latter is cloned between the XhoI and BamHIsites of pTG2673. The vector pTG6355 is obtained (FIG. 5).

Viral particles are generated by transfection of the GP+E-86 cells withthe vectors pTG6355, pTG6356 and pTG6357 (cotransfection with pLXSP)according to the technique described in Example 1, the only differencebeing that the transfected clones are tested for the production of17-1-A antibodies as indicated below. 100 μl of anti-murineimmunoglobulin antibody (Southern Biotechnology) previously diluted 100fold in a buffer (80 mM Na₂CO₃, 200 mM NaHCO₃, pH9.6) are distributed inthe wells of a microtiter plate (Nunc) and incubated overnight at 4° C.The nonadsorbed antibodies are removed by extensive washes with 1×PBSbuffer, 10 mM EDTA, 0.05% Tween 20. The wells are saturated by additionof 200 μl of a 1×PBS solution, 1% BSA (Bovine Serum Albumin) 1 h at 37°C. After this stage, the plate is again washed, and then the controlseries (diluted in 1×PBS, 1% BSA) or the culture supernatants to betested are deposited and incubated for 2 h at room temperature, withagitation. After several washes, the plates are incubated with 100 μl ofan anti-Ig₂a goat antibody (isotype of 17-1-A), coupled to biotin(Southern Biotechnology) diluted 5000 fold in 1×PBS buffer, 1% BSA.After 1 h of incubation at room temperature with agitation, the excessis removed by 8 washes and then 100 μl of a peroxidase-streptavidinsolution (Amersham), diluted 1000 fold, are distributed. Afterincubation for 45 min followed by extensive washes, the enzymaticactivity is revealed by addition of 100 μl of substrate solution (forone plate: 12.5 ml of 25 mM citrate buffer, 50 mM Na₂HPO₄, pH 5; 1pastille of 5 mg of OPD (ortho-phenoldiamine, Sigma); 5 μl of 35% H₂O₂).The reaction is stopped by addition of 25 μl of 3M H₂SO₄ per well. Theabsorbance is then read at 490 nm.

The quantity of antibody present in the culture supernatants isestablished as a function of a calibration series prepared as follows:the 17-1-A hybridoma cells are injected into “nude” mice. Afterformation of ascites, the ascitic fluid is collected and the 17-1-Aantibody is purified therefrom by passage on a protein A sepharosecolumn. This is a conventional technique accessible to persons skilledin the art. A solution of purified 17-1-A antibody is prepared at aconcentration of 1 μg/ml and then 2-fold serially diluted in a PBSbuffer. The absorbance is measured for each of the dilutions and thecalibration curve is established (ng of antibody as a function of theabsorbance). The most productive clones secrete, according to thetransfected vector, from 200 to 900 ng of 17-1-A antibody/10⁶ cells/24h.

B. Preparation of the Implant

The NIH3T3 cells are infected with the most productive clones (derivedfrom the transfection of the GP+E-86 cells with the vectors pTG6355,pTG6356 and pTG6357) and the culture supernatants tested by ELISA forthe secretion of 17-1-A antibody. An antibody level varying from 100 to1000 ng/10⁶ cells/24 hours is detected according to the constructs.

Moreover, it is checked that the antibody produced recognizes the GA733antigen expressed by the SW948 cells. For that, the flow cytometrytechnique is used. The SW948 cells (ATCC CCL237) are cultured incomplete DMEM medium. They are detached by the action of trypsin,counted and then 5×10⁵ cells are distributed in wells of a 96-wellplate. This plate is centrifuged for 1 min at 1000 rpm, without brakes,to pellet the cells. The supernatant is removed and then the plate isvortexed and the cells resuspended in 100 μl of FACS buffer (cationic1×PBS, 1% BSA, 0.1% human γ globulins, 5 mM EDTA). The plate is againcentrifuged and the supernatant removed. The cells are then resuspendedin the culture supernatant to be tested or in a dilution of the controlantibody in FACS buffer and incubated for 1 h at 4° C. 4 washes are thencarried out under the conditions described above and then the cells areresuspended in 100 μl of a fluorescein-coupled anti-mouse immunoglobulingoat F(ab′)₂ fragment (DTAF) (Jackson Immuno Research Laboratories)diluted 100 fold in FACS buffer. Another incubation of 1 h at 4° C.allows the antibody to bind. The excess is then removed by 4 washes andthe cells are finally resuspended in 300 μl of cationic 1×PBS beforebeing analyzed with a flow cytometer FACScan (Becton Dickinson). It isobserved that all the NIH3T3 supernatants tested (resulting from theinfection with 3 types of viral particles) secrete an antibody capableof binding to the target GA733 protein.

In the case of the constructs where the antibody is fused to barnase orto the attenuated version thereof (pTG6356 and pTG6357), it isadvantageous to be able to check that the fused antibody has a nucleaseactivity. For that, the degradation of a tRNA is monitored. 100 μl ofsupernatants to be tested or of an RNAseA solution at a knownconcentration (as reaction control) are added to 200 μl of 0.5 MTris-HCl, pH 7.5, 5 mM EDTA, 0.5 mg/mg of BSA and 1 mg/ml final of tRNAand incubated at 37° C. for 30 min. The tubes are then placed on ice and700 μl of 6% perchloric acid are added to precipitate the tRNA for 10min on ice. A 10 min centrifugation at a maximum speed at 4° C. makes itpossible to pellet the tRNA, leaving in suspension the free nucleotidesreleased by the action of the enzyme. The absorbance of thesenucleotides is then read at 260 nm.

The implant may be constituted according to the method indicated inExample 1 by incorporation of primary murine fibroblasts or of NIH3T3cells, transduced with the vectors pTG6355, pTG6356 or pTG6357.

Example 3 Preparation of an Implant Secreting an Immunoadhesin andIntended for an Anti-AIDS Immunotherapy

The aim is to produce an immunoadhesin resulting from the fusion of an“adhesive” molecule binding the HIV virus glycoprotein and of animmunoglobulin stabilizing the structure and conferring a nonspecificimmunity. The adhesive part is derived from the CD4 membrane protein(structure represented in FIG. 6) from which the N-terminal part isretained (signal sequence and I and II domains of the extracellularregion). Several studies have shown that they are capable, bythemselves, of binding gp120, of blocking the interaction and thepenetration of HIV into CD4^(+ target cells (Traunecker et al.,) 1988,Nature 331, 84-86; Deen et al., 1988, Nature 331, 82-84; Hussey et al.,1988, Nature 331, 78-81; Fisher et al., 1988, Nature 331, 76-78). Theimmunoglobulin part consists of the constant γ3 region (hinge region—CH2-CH3) of the 2F5 antibody. The hybrid molecule is subsequentlydesignated sCD4-2F5.

The construction is carried out in the following manner (see FIG. 7):

The sequences encoding the sCD4 region (signal sequence—I and IIdomains) are conventionally isolated by PCR. As template, there is usedthe cDNA CD4 described in the literature (obtained from mRNA ofCD4^(+ cells) or a prior art plasmid in which the cDNA is cloned (Jay Maddon et al.,)1985, Cell 42, 93-104), which is hybridized with the primers OTG7094 andOTG7095 (SEQ ID NO: 13 and 14). The first makes it possible to introducean XhoI site and Kozak type consensus sequences upstream of the CD4initiator ATG and the second carries nucleotides corresponding, on theone hand, to the C-terminal end of the II CD4 domain and, on the otherhand, to the N-terminal end of the hinge region of the 2F5 HC. Thereaction occurs over 25 cycles (1 min at 94° C., 2 min at 50° C. and 3min at 72° C.).

The sequences encoding the constant γ3 segment of the 2F5 HC are alsoamplified by PCR. The plasmid pTG2677 and the primers OTG7097 andOTG7096 (SEQ ID NO: 15 and 16) are used. The first is complementary toOTG7095 and the second covers the XmaI site situated within the CH3region.

The PCR products thus generated overlap over 30 bp. They arerehybridized and subjected to a second amplification reaction whichoccurs in 2 stages, in the first instance, a linear amplification toextend the rehybridized product (10 cycles: 1 min at 94° C., 2 min at37° C. and 3 min at 72° C.) followed by an exponential amplification inthe presence of the primers OTG7094 and OTG7096 (SEQ ID NO: 13 and 16)(20 cycles: 1 min at 94° C., 2 min at 50° C. and 3 min at 72° C.).

The final product is inserted between the XhoI and XmaI sites of pTG2677to give pTG8332 in order to reconstitute the complete sCD4-2F5 molecule.The latter is excised by XhoI-BamHI digestion and cloned downstream ofthe murine PGK promoter into the vector pTG6368. pTG8338 is obtained(FIG. 8).

The NIH3T3 cells are transfected with 10 μg of BglII-linearized pTG8338.Two days later, the cells are cultured in an increasing puromycinconcentration (5 to 75 μg/ml). The expression of the sCD4-2F5 protein isverified by immunofluorescence using an antibody which recognizes eitherthe CD4 part (Leu3A; Becton Dickinson) or the 2F5 part (mouse monoclonalantibody to the hinge region of a human IgG3; Interchim) and a conjugateconsisting of a fluorescein-coupled anti-mouse Ig donkey antibody(Jackson Laboratories). It is noted that about 30% of the cellular poolexpresses a detectable level of immunoadhesin. For this reason,producing clones are isolated by clonal dilution.

The production of the viral particles is carried out in transfectedGP+E-86 cells according to the conventional procedure and selected inthe presence of puromycin. The target NIH3T3 cells are then infectedwith the cell supernatant and the immunofluorescence analyses confirmthe expression of the transgene in 100% of the cells.

The quantification is performed by ELISA. In the first place, 500 ng ofHIV-1 virus gp160 envelope glycoprotein are deposited so as to bind theCD4 part of the CD4-2F5 molecule. It is produced by the recombinantroute as indicated in international application WO 92/19742. Thesupernatant to be assayed is then added and, finally, an antibodydirected against the 2F5 part (peroxidase-conjugated anti-human Ig goatantibody; Interchim). The calibration solution consists of therecombinant immunoadhesin purified from the culture supernatants on afast-flow sepharose-G protein column (Pharmacia). A productivity greaterthan 10 μg/ml/24 h/10⁶ cells is measured on the supernatants of theinfected NIH 3T3 cells. This test also indicates that the protein iscapable of binding gp160 and, consequently, would be capable of bindingthe HIV virus in order to exert its therapeutic function.

The infected NIH3T3 cells are amplified by culturing in F175 flasks. 4organoids each containing about 10⁷ cells are generated by applying thetechnology detailed in Example 1 and transplanted in the peritonealcavity of 4 nude BALB/c female mice. The secretion of immunoadhesin intothe serum is monitored up to 5 weeks post implantation (assay by ELISA).The results indicate a concentration of the order of 100 to 200 μg/mland a continuous secretion during the duration of the experiment.

Example 4 Preparation of an Implant Secreting a Protein Resulting fromthe Fusion of the Immunoadhesin sCD4-2F5 and of the Human Angiogenin

Angiogenin is a 14.1 kDa plasma protein belonging to the family ofribonucleolytic enzymes. However, its lytic action is more limited thanthat of the reference ribonuclease A (RNase A) and shows a markedpreference for certain RNAs (especially the 18S and 28S ribosomal RNAsand the transfer RNAs). The gene and the cDNA were cloned about tenyears ago (Kurachi et al., 1985, Biochemistry 74, 5494-5499).

The fusion of the sequences encoding angiogenin downstream of sCD4-2F5should allow the synthesis of a protein capable of targeting anddestroying the cells infected with HIV. They were obtained by PCR fromthe plasmid pHAG1 (Kurachi et al., 1985, supra) with the aid of theprimers OTG10089 and OTG100090 (SEQ ID NO: 17 and 18) comprising attheir 5′ ends the EcoRI and BamHI restriction sites situated in 5′ ofthe first codon of the mature protein and in 3′ of the stop codon,respectively.

For reasons of stearic hindrance, it is chosen to introduce a spacerbetween the two entities. The PCR reaction is carried out with the aidof the template pTG2677 and the oligonucleotides OTG10087 and OTG10088(SEQ ID NO: 19 and 20) in order to amplify the part of the 2F5 genestretching from the XmaI site (inside CH3) to the stop codon. The primerOTG10088 is designed to eliminate the stop codon and introduce in 3′ aBamHI site as well as the spacer encoding the residuesGly-Gly-Gly-Gly-Ser (SEQ ID NO 22).

The two PCR fragments obtained, bordered by a BamHI site are ligatedtogether. The XmaI-EcoRI fragment is isolated and inserted, first intopBluescript in order to verify the sequence and, finally into the vectorpTG8332 in order to reconstitute the complete fusion sequence“sCD4-2F5-Angiogenin”. pTG8373 is obtained (FIG. 9). The complete unitmay be excised by XhoI-BglII digestion and cloned into the retroviralvector pTG6368 linearized with XhoI and BamHI. The viral particles maybe constituted as above and the organoids generated from infected targetcells NIH3T3 or primary fibroblasts.

Example 5 Preparation of an Adenoviral Vector Expressing theImmunoadhesin sCD4-2F5

The adenoviral genome fragments used in the constructions describedbelow are indicated precisely according to their position in thenucleotide sequence of the type 5 adenovirus (Ad5) genome as disclosedin the Genebank data bank under the reference M73260.

The intron and the polyadenylation signal (pA) of the β-globin humangene are obtained from the vector pBCMG/Neo (Karasuyama, 1988, Eur J.Immuno. 18, 97-104; Karasuyama, 1989, J. Exp. Med. 169, 13-25) andintroduced into the plasmid pREP4 (InVitogen™) downstream of the RSVvirus 3′ LTR. The cassette “RSV-intron-pA β-globin promoter” is isolatedfrom the preceding vector in the form of a SalI-BamHI fragment andinserted into the vector pTG9350. The latter is obtained from thecloning of the Ad5 genomic sequences stretching from nucleotides 1 to458 and 3328 to 5788 in p polyII (Lathe et al., 1987, Gene 57, 193-201).

A polylinker provided with multiple cloning sites (EcoRI, XhoI, NotI,XbaI, SpeI, BamHI, EcoRV, HindIII, ClaI, KpnI and BglII) is introducedinto the vector pTG8346 obtained in the preceding stage (between theintron and pA), to create pTG8347.

The sequences encoding the hybrid protein sCD4-2F5 are isolated from thevector pTG8338 (Example 3) by XhoI-BamHI digestion and introduced intothe vector pTG8347 cleaved with XhoI and BglII1, to generate pTG8349which allows their expression under the control of the RSV promoter.

The in vitro homologous recombination technique (described in FrenchApplication 94 14470) is used to reconstitute the complete genome of therecombinant vector. There is used to this end the vector pTG4656 whichcomprises the Ad5 genome deleted of the E1 and E3 regions (Ad5 1 to458-Ad2 MLP promoter-LacZ gene-pA SV40-Ad5 3329 to 28529 and 30470 to35935). Any other E1⁻ E3⁻ adenoviral vector may also be suitable, suchas those described in international application WO 94/28152. The BJ5183cells (Hanahan, 1983, J. Mol. Biol. 166, 557-580) are cotransformed bypTG4656 linearized with the enzyme ClaI (10 to 20 ng) and the PacI-BstXIfragment purified from pTG8349 (about 10-fold molar excess). Therecombination at the level of the homologous adenoviral sequences causesthe replacement of the LacZ cassette of pTG4656 by that of sCD4-2F5 (RSVpromoter-β-globin intron-sCD4-2F5 gene-pA β-globin) carried the fragmentderived from pTG8349. The vector pTG8357 is generated (FIG. 10).

The recombinant viruses are obtained by transfection of pTG8357 into thecells 293 (ATCC CRL1573). 5 plaques are selected which are amplified byculturing in F25 flasks in the presence of fresh 293 cells. After 5days, the infected cells are harvested and subjected to a HIRTH analysis(Gluzman and Van Doren, 1983, J. Virol. 45, 91-103), in order to verifythe presence of the transgene. Briefly, the HIRTH analysis consists ofan extraction of the adenoviral genome, a precipitation of the viralDNA, a digestion with an appropriate restriction enzyme, a transfer ontoa membrane and a hybridization with a radioactive probe capable ofhybridizing with the sCD4-2F5 sequences. A positive signal is observedfor the 5 adenoviruses analyzed.

A substantial adenoviral stock is constituted by successiveamplification in the 293 cells, purification on two cesium chloridegradients and dialyses. This stock may be used in the context ofclinical anti-AIDS tests.

Example 6 Preparation of an Adenoviral Vector Expressing the CytotoxicImmunoadhesin sCD4-2F5-Angiogenin

The sequences encoding the fusion protein sCD4-2F5-angiogenin areexcised from the vector pTG8373 (Example 4) by XhoI-BglII digestion andcloned at the level of the same sites in the vector pTG8347 (Example 5).The vector pTG8376 is obtained (FIG. 11) which may be subjected tohomologous recombination with an adenoviral vector, for example pTG4656,to produce the defective recombinant adenoviruses expressing thecytotoxic immunoadhesin directed against the cells infected with the HIVvirus.

1. Implant of genetically modified cells comprising an exogenousnucleotide sequence encoding all or part of an antibody, said exogenousnucleotide sequence being placed under the control of the elementsnecessary for its expression and for the secretion of said antibody. 2.Implant according to claim 1, wherein said antibody is selected from thegroup consisting of: a native antibody, a chimeric antibody, an antibodyfragment, especially a fragment Fab, F(ab′)₂, Fc or scFv, and abispecific antibody.
 3. Implant according to claim 1, wherein saidantibody is modified by a toxic or immunopotentiating substance. 4.Implant according to claim 3, wherein said antibody may be modified by atoxic substance selected from a ribonuclease, and especially theribonuclease from Bacillus amyloliquefaciens, ricin, diphtheria toxin,cholera toxin, herpes simplex virus thymidine kinase, cytosine deaminasefrom Escherichia coli or from a yeast of the genus Saccharomyces,exotoxin from Pseudomonas and human angiogenin or an analog of saidsubstances.
 5. Implant according to claim 1, wherein cells aregenetically modified by transfection of a vector derived from a plasmid,from a retrovirus or from a herpes virus, from an adenovirus, from anadenovirus-associated virus comprising said exogenous nucleotidesequence placed under the control of the elements necessary for itsexpression and for the secretion of the said antibody.
 6. Implantaccording to claim 5, wherein said vector is dicistronic.
 7. Implantaccording to claim 6, wherein said vector is retroviral and comprisesfrom 5′ to 3′: (a) a 5′ LTR derived from a retrovirus, (b) anencapsidation region, (c) an exogenous nucleotide sequence comprising:an internal promoter, a first sequence encoding the heavy chain of anantibody, a ribosome entry initiation site, a second sequence encodingthe light chain of an antibody, and (d) a 3′ LTR derived from aretrovirus.
 8. Implant according to claim 7, wherein said exogenousnucleotide sequence comprises, in addition, a third sequence encoding atoxic or immunopotentiating substance fused downstream and operably tothe second sequence.
 9. Implant according to claim 1, comprisinggenetically modified autologous cells.
 10. Implant according to claim 9,comprising genetically modified fibroblasts.
 11. Implant according toclaim 1, wherein from 10⁶ to 10¹², preferably from 10⁷ to 10¹¹genetically modified cells.
 12. Method for the preparation of an implantaccording to claim 1, wherein the genetically modified cells and anextracellular matrix are placed in contact.
 13. A pharmaceuticalcomposition intended for the treatment or for the prevention of anacquired disease comprising the implant according to claim
 1. 14. Apharmaceutical composition intended for the treatment or for theprevention of an infectious disease cancer comprising the implantaccording claim
 1. 15. Recombinant adenoviral vector comprising anexogenous nucleotide sequence encoding all or part of an antibody andplaced under the control of the elements necessary for its expression,wherein said antibody is modified by a toxic or immunopotentiatingsubstance.
 16. Recombinant adenoviral vector according to claim 15, thewherein said antibody is selected from the group consisting of a nativeantibody, a chimeric antibody, an antibody fragment and especially afragment F(ab′)₂, Fc or scFv and a bispecific antibody.
 17. Recombinantadenoviral vector according to claim 15, wherein said antibody may bemodified by a toxic substance selected from a ribonuclease, andespecially the ribonuclease from Bacillus amyloliquefacienis, ricin,diphtheria toxin, cholera toxin, herpes simplex virus thymidine kinase,cytosine deaminase from Escberichia coli or from a yeast of the genusSaccharomyces, exotoxin from Pseudomonas and human angiogenin or ananalog of said substances.
 18. Recombinant adenoviral vector accordingto claim 15, wherein said antibody is modified by an immunopotentiatingsubstance.
 19. Recombinant adenoviral vector comprising an exogenousnucleotide sequence encoding all or part of one or more protein(s) ofinterest capable of forming a multimer, such as a dimer or a tetramer,in a host cell; the said exogenous nucleotide sequence being placedunder the control of the elements necessary for its expression, saidvector being derived from an adenovirus of human, canine, avian, bovine,murine, ovine, porcine or simian origin or a hybrid comprisingadenoviral genome fragments of different origins.
 20. Recombinantadenoviral vector according to claim 15, derived from an adenovirus ofhuman, canine, avian, bovine, murine, ovine, porcine or simian origin orfrom a hybrid comprising adenoviral genome fragments of differentorigins.
 21. Recombinant adenoviral vector according to claim 15,wherein it is defective for replication.
 22. Recombinant adenoviralvector according to claim 21, wherein it at least lacks all or part ofthe E1 region and, optionally, all or part of the E3 region. 23.Recombinant adenoviral vector according to claim 21, comprising anexogenous nucleotide sequence encoding the heavy chain of the 2F5antibody, an IRES element and the light chain of the 2F5 antibody; saidexogenous nucleotide sequence being placed under the control of theelements necessary for its expression.
 24. Recombinant adenoviral vectoraccording to claim 21, comprising an exogenous nucleotide sequenceencoding the signal sequence and the extracellular I and II domains ofthe CD4 protein operably fused to the constant γ3 region (hingeregion-CH2 and CH3) of the heavy chain of the 2F5 antibody. 25.Recombinant adenoviral vector according to claim 21, comprising anexogenous nucleotide sequence encoding the signal sequence and theextracellular I and II domains of the CD4 protein operably fused to theconstant γ3 region (hinge region-CR2 and CR3) of the heavy chain of the2F5 antibody and operably fused to the mature human angiogenin. 26.Recombinant adenoviral vector according to claim 15, wherein theelements necessary for the expression comprise a promoter selected fromthe group consisting of the adenoviral early promoter E1A, the latepromoter MLP (Major Late Promoter), the murine or human PGK(Phosphoglycerate kinase) promoter, the SV40 virus early promoter, theRSV (Rous Sarcoma virus) virus promoter, a promoter which isspecifically active in tumor cells and finally a promoter which isspecifically active in the infected cells.
 27. Infectious viral particlecomprising a recombinant adenoviral vector according to claim
 15. 28.Eukaryotic host cell comprising a recombinant adenoviral vectoraccording to claim
 15. 29. Pharmaceutical composition comprising arecombinant adenoviral vector according to claim 15, in association witha pharmaceutically acceptable carrier.
 30. Pharmaceutical compositionaccording to claim 29, comprising 10⁴ to 10¹⁴ pfu.
 31. Pharmaceuticalcomposition according to claim 29 wherein it is in injectable form. 32.A method for the preparation of a pharmaceutical composition intendedfor the treatment and/or prevention of the human or animal body by genetherapy comprising using the recombinant adenoviral vector according toclaim
 15. 33. The method according to claim 32, wherein saidpharmaceutical composition is intended for the treatment and/orprevention of acquired diseases and especially cancers and AIDS.
 34. Themethod according to claim 33, wherein said pharmaceutical compositionadministrable via the intravenous or intratumor route.